Genomics to detect transmission of livestock-associated methicillin-resistant Staphylococcus aureus from UK pigs in abattoirs during slaughter.

BACKGROUND: Livestock-associated MRSA (LA-MRSA) transmission/cross-contamination can occur at abattoir through colonized pigs, increasing occupational hazards and health concerns for workers. To assess this risk we used genomics to identify LA-MRSA lineages present in batches of pigs sent to slaughter and distribution of clones. METHODS: WGS was performed on 85 LA-MRSA previously isolated from six abattoirs from 105 batches of pigs sent from 100 UK farms. spa typing and MLST were performed on all isolates. A mashtree tree was constructed to compare genomes of the LA-MRSA with 1281 global isolates from livestock and humans. A phylogenetic tree and pairwise SNP distance matrices were built from whole genomes of 109 isolates closest to those from abattoirs to compare evolutionary relationships and identify clones. RESULTS: All abattoir isolates belonged to CC398 and were mainly of spa type t011, although other spa types were present. Phylogenetic analysis confirmed the abattoir isolates were most closely related to each other and to pig LA-MRSA from across Europe, indicating a common evolutionary origin with related lineages colonizing UK pigs.Comparison of genomes using SNPs suggested between one and four clones were transferring between pigs from different batches. Transmission likely occurred on farm premises, during transportation, and/or within abattoirs through contact with contaminated surfaces in lairage or post-stunning. CONCLUSIONS: Genomics forensically identified related isolates/clones circulating in pigs at slaughter, showing contamination occurs often. Results suggest that further genomic tracking will identify hotspots, and improvements in measures such as biosecurity and disinfection will help reduce risk for workers.

Numerical and experimental investigation of static wetting morphologies of aqueous drops on lubricated slippery surfaces using a quasi-static approach.

The static wetting behavior of drops on surfaces with thin lubricating films is very different compared to solid surfaces. Due to the slow dynamics of the wetting ridge, it is challenging to predict the apparent contact angles of such drops. It is hypothesized that for a sinking drop on a lubricated surface, quasi-static wetting morphology can be numerically computed from the knowledge of interfacial energies, lubricant thickness, and drop volume. In this study, we use Surface Evolver to numerically compute the static wetting morphology for the four-phase system using a quasi-static approach with a sinking time similar to the early-intermediate times, and the results agree well with the corresponding experiments. We find that the apparent contact angles depend significantly on the lubricant thickness and substrate wettability compared to other parameters.

Dewetting of non-polar thin lubricating films underneath polar liquid drops on slippery surfaces.

HYPOTHESIS: The stability of thin lubricating fluid-coated slippery surfaces depends on the surface energy of the underlying solid surface. High energy solid surfaces coated with thin lubricating oil lead to the dewetting of the oil films upon depositing aqueous drops on them. Hence such surfaces are very suitable to investigate dewetting of thick films (thickness > 500 nm), which otherwise is not possible using a conventional dewetting system. EXPERIMENTS: Lubricating films of different thicknesses are coated on hydrophilic solid surfaces, and glycerol drops are deposited on them. Fluorescence imaging of lubricating films and macroscopic wetting behavior of glycerol drops are analyzed to understand the dewetting phenomenon. FINDINGS: Underneath lubricating films undergo initial thinning and subsequently dewet. The dewetting dynamics during hole nucleation and growth and the final pattern of the dewetted oil droplets depend strongly on the thickness of the lubricating films. Ultrathin films dewet spontaneously via homogeneous nucleation, whereas thicker films dewet via heterogeneous nucleation. During dewetting, the apparent contact angle and radius of glycerol drops follow universal scaling behavior.

Evaporation dynamics of pure and binary mixture drops on dry and lubricant coated slippery surfaces.

HYPOTHESIS: Lubricating fluid coated slippery (LCS) surfaces offer a new scope to study the evaporation of sessile drops due to pinning free motion of the three-phase contact line (TPCL). This work aims to experimentally demonstrate the different evaporation behavior of water and binary mixture drops on dry and LCS surfaces. EXPERIMENTS: Evaporation study on dry and LCS surfaces is performed by capturing top and side views of evaporating drops to extract various parameters which are subsequently used to distinguish between different evaporation modes. FINDINGS: Formation of a wetting ridge and cloaking of water drops on LCS surfaces affect the overall evaporation process and make it different compared to that on dry surfaces. Evaporation dynamics on LCS surfaces reveal that wetting ridge height of an evaporating drop varies non-monotonically compared to the drop height. Diffusion based theoretical model is used to predict the role of various system parameters on the evaporation process. In contrast to dry solid surfaces, where coffee ring effects are commonly observed towards the end of the evaporation process, LCS surfaces show the formation of a wrinkle like pattern of the lubricating fluid which disappears at long times.

Mobility of Aqueous and Binary Mixture Drops on Lubricating Fluid-Coated Slippery Surfaces.

The mobility of liquid drops on lubricant-infused slippery surfaces depends strongly on various system parameters, for example, surface energy and roughness of the underlying solid surface and surface tension and viscosity of the test and the lubricating fluids. Here, we investigate lubricant-coated slippery surfaces fabricated on smooth hydrophobic solid surfaces and examine the influence of thickness and viscosity of the lubricating oil on the velocity of aqueous drops. We also investigate the effect of surface tension of the test liquid using a binary mixture of water and ethanol, on the apparent contact angle, which further affects their slip velocity. A theoretical model, based on various dissipative forces acting in different regions of the lubricating oil and a test drop, is also presented, which elucidates the dependence of drop velocity on lubricating oil viscosity and base radius of drops of test liquids.

A rare case of acyanotic congenital heart disease, large patent ductus arteriosus with pre-ductal coarctation of descending thoracic aorta with patent ductus arteriosus closure and extra anatomical bypass grafting.

We report a case of 18-year-old female patient with large patent ductus arteriosus (PDA)-preductal coarctation of descending thoracic aorta. She underwent large PDA closure with a prosthetic graft from ascending aorta to descending thoracic aorta by mid-sternotomy on cardiopulmonary bypass machine under total hypothermic circulatory arrest.

Amyloidosis and Anesthesia.

AIM: The aim of this article is to provide a view of amyloidosis and discuss implications for the anesthetic management of patients with this condition. MATERIAL AND METHOD: Urine samples from patients with plasma cell dyscrasias were obtained from a urine bank that gathers urine samples from patients who gave research use consent for specimens that would otherwise be considered waste. RESULTS: Patients with amyloidosis may present to the anesthesiologist for procedures relating to diagnosis, surgery relating to the underlying condition (e.g., bronchial laser and organ transplant), or for incidental surgery. The condition carries a significant risk of perioperative morbidity and mortality. CONCLUSION: The term amyloid was coined by Virchow in the mid 19th century, meaning "starch like." Amyloidosis is a disease complex, in which there is an abnormal deposition of extracellular hyaline material with particular staining characteristics and which contains protein fibrils embedded in a relatively amorphous ground substance. There are numerous clinical manifestations, the onset is insidious, and the diagnosis may not be made in a patient undergoing anesthesia and surgery for an apparently straightforward problem. Unexpected complications such as heart or kidney failure may arise, either before operation or in the postoperative period. Bullous lesions of the skin or oral mucosa and extensive areas of purpura are but two of the ways, in which amyloidosis may first present. The disease spectrum may be inherited or acquired, localized or systemic, and life threatening or an incidental finding.

Livestock-Associated Methicillin Resistant Staphylococcus aureus (LA-MRSA) Clonal Complex (CC) 398 Isolated from UK Animals belong to European Lineages.

In recent years, there has been an increase in the number of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 recovered from S. aureus isolated animals in the UK. To determine possible origins of 12 LA-MRSA CC398 isolates collected after screening more than a thousand S. aureus animal isolates from the UK between 2013 and 2015, whole genome sequences (WGS) of CC398 European, including UK, and non-European isolates from diverse animal hosts were compared. Phylogenetic reconstruction applied to WGS data to assess genetic relatedness of all 89 isolates, clustered the 12 UK CC398 LA-MRSA within the European sub-lineages, although on different nodes; implicating multiple independent incursions into the UK, as opposed to a single introduction followed by clonal expansion. Three UK isolates from healthy pigs and one from turkey clustered within the cassette chromosome recombinases ccr C S. aureus protein A (spa)-type t011 European sub-lineage and three UK isolates from horses within the ccrA2B2 t011 European sub-lineage. The remaining UK isolates, mostly from pigs, clustered within the t034 European lineage. Presence of virulence, antimicrobial (AMR), heavy metal (HMR), and disinfectant (DR) resistance genes were determined using an in-house pipeline. Most, including UK isolates, harbored resistance genes to ≥3 antimicrobial classes in addition to ß-lactams. HMR genes were detected in most European ccrC positive isolates, with >80% harboring czrC, encoding zinc and cadmium resistance; in contrast ~60% ccrC isolates within non-European lineages and 6% ccrA2B2 isolates showed this characteristic. The UK turkey MRSA isolate did not harbor φAVß avian prophage genes (SAAV_2008 and SAAV_2009) present in US MSSA isolates from turkey and pigs. Absence of some of the major human-associated MRSA toxigenic and virulence genes in the UK LA-MRSA animal isolates was not unexpected. Therefore, we can conclude that the 12 UK LA-MRSA isolates collected in the past 2 years most likely represent separate incursions into the UK from other European countries. The presence of zinc and cadmium resistance in all nine food animal isolates (pig and poultry), which was absent from the 3 horse isolates may suggest heavy metal use/exposure has a possible role in selection of some MRSA.

Detection of antibiotic residues and association of cefquinome residues with the occurrence of Extended-Spectrum ß-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011.

Waste milk samples from 103 farms in England and Wales were examined for the presence of ß-lactam antibiotics and ESBL-producing Enterobacteriaceae. Approximately 10 months after the initial sampling, further waste milk, environmental and faecal samples from farms shown to be positive for CTX-M Escherichia coli were investigated further. Isolates with an ESBL phenotype were tested by PCR for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes. Isolates positive for blaCTX-M were sequenced to determine CTX-M type. Representative isolates were further examined by PFGE, plasmid replicon typing and serotyping. Of particular interest, 21.4% of waste milk samples contained residues of the cephalosporin cefquinome, which was significantly associated with CTX-M bacteria. Such bacteria occurred in 5.8% of the waste milk samples (including 3.9% CTX-M E. coli). CTX-M types identified were 1, 14, 14b and 15, but none of the E. coli were serotype O25, the serotype of the human pandemic strain.

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum ß-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla(CTX-M-group-1) dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla(CTX-M-group-1) and bla(OXA-1-like) genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6')-Ib, catB3, bla(OXA-1-like) and bla(CTX-M-group-1). forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Real-time PCR approach for detection of environmental sources of Campylobacter strains colonizing broiler flocks.

Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 10(2) to 3.7 x 10(2) CFU/ml in bacterial cultures and 6.6 x 10(2) CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.